Smoking is a behavior that results from a complex developmental interplay of genetic and environmental processes. Smoking, in turn, causes a number of adverse health outcomes. Using other tissue preparations, we and others have shown that some of these adverse effects of smoking may be mediated by altered DNA methylation. However, whether smoking induces differential DNA methylation in lymphocytes is still highly controversial and whether any observed differential methylation is functionally relevant is completely unknown. In this R21 application, we propose to unequivocally determine whether smoking is associated with changes in lymphocyte DNA methylation and set the stage for future investigations by ourselves and others. To accomplish this goal, we will use the bioresources of two large, informative, ethnically diverse populations. The first population, the Iowa Adoption Studies (IAS) is the largest case and control adoption study in the United States. The IAS has been pivotal in the demonstration of the importance of genetic (G), environmental (E) factors and gene-environment (GxE) interactions in the development and maintenance of common behavioral illnesses including major depression and nicotine dependence. The second population, the Family and Community Health Studies (FACHS), is a large longitudinal study of the impact of sociopsychological factors such as racism and poverty on depression and health behaviors in 900 rural African-American families. The over-arching hypotheses of our research group are that substance use is associated with epigenetic changes and that some of these changes may be important in both moderating the continuation of substance use and the vulnerability to smoking related comorbidities. Unfortunately, to date, many in the field do not believe that smoking, or any other form of substance use, leads to alterations in peripheral lymphocyte methylation. To set the groundwork for integrated studies of the role of epigenetic factors in smoking, we will test the hypothesis that smoking is associated with changes in DNA methylation. To do this, we will use our established approaches to identify genomic regions whose methylation is altered in smoking using primary lymphocyte DNA from female IAS and 800 FACHS subjects. Because our populations are well characterized for behavioral and physical outcomes, we have complete sets of biomaterials and data for future studies and are embedded transdisciplinary collaboration, we are well poised to rapidly exploit any positive findings. This application is innovative because the effect of smoking on lymphocyte DNA methylation is highly controversial and has not been unequivocally demonstrated. Since lymphocytes are key players in smoking associated autoimmune illnesses and may be effective proxies for CNS epigenetic processes, the successful demonstration of effects of smoking on DNA methylation will have an impact on health care because these investigations of this critical cell type may lead to new interventional strategies for smoking and smoking related comorbidities. It is highly feasible because it builds off existing populations and repositories. Finally, the investigative team is well prepared and includes a molecular biologist/psychiatrist with extensive experience in methylation studies and a well-trained bioinformatician.